Summary:[unreadable] [unreadable] Burkholderia pseudomallei, the etiological agent of melioidosis, is a Gram-negative, facultatively anaerobic, motile bacillus that is responsible for a broad spectrum of illnesses observed in both humans and animals. Burkholderia mallei, the etiological agent of glanders, is a Gram-negative bacterium that is responsible for disease in donkeys, mules, horses and occasionally humans. Unlike the environmental saprophyte B. pseudomallei, however, B. mallei does not persist in nature outside of its soliped hosts. While B. mallei and B. pseudomallei are genotypically similar, significant phenotypic differences do exist between the two pathogenic species. Although glanders is one of the oldest diseases known to man, relatively little is known about the pathogenesis of disease caused by B. mallei. This phenomenon is primarily due to the lack of disease in North America along with the fact that B. mallei can be a particularly dangerous organism to study even in a controlled laboratory environment. Both bacteria are considered BL3 select agents by the CDC.[unreadable] [unreadable] Characterization of the lipid A and O-antigen moities of the LPS of B. pseudomallei and B. mallei: [unreadable] [unreadable] Lipopolysaccharides (LPS), also commonly referred to as endotoxins, are a major component of Gram-negative cell envelopes. The barrier functions provided by bacterial outer membranes are largely due to the presence of these molecules. LPS antigens expressed by smooth strains are composed of three covalently linked domains: an O-antigen, a core region and a lipid A moiety. O-antigens, consisting of oligosaccharide repeats, are the outermost domains of LPS molecules expressed on bacterial cell surfaces. Because of this, they are often a primary target of innate and acquired immune responses. Lipid A, the hydrophobic membrane-anchor component of LPS molecules, is the domain responsible for stimulating pathophysiological responses in mammals such as cytokine production, inflammation and shock. [unreadable] [unreadable] To better understand the role of lipopolysaccharide (LPS) in the pathogenesis of these diseases, studies were initiated to characterize the structural and biological properties of lipid A moieties expressed by this organism. Using a combination of chemical analyses and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, B. mallei was shown to express a heterogeneous mixture of tetra- and penta-acylated lipid A species that were non-stoichiometrically substituted with 4-amino-4-deoxy-arabinose residues. The major penta-acylated species consisted of bisphosphorylated D-glucosamine disaccharide backbones possessing two amide linked 3-hydroxyhexadecanoic acids, two ester linked 3-hydroxytetradecanoic acids (C14:0(3-OH)) and an acyloxyacyl linked tetradecanoic acid, whereas, the major tetra-acylated species possessed all but the 3-linked C14:0(3-OH) residues. In addition, studies demonstrated that B. mallei LPS was able to stimulate human embryonic kidney 293 cells expressing human Toll-like receptor 4 (hTLR4) complexes to secrete high levels of IL-8. Furthermore, when stimulated with B. mallei LPS, human macrophage-like cells (THP-1 and U-937) as well as monocyte-derived macrophages and dendritic cells secreted TNF-, IL-6 and RANTES at levels similar to those observed using Escherichia coli LPS as a control. Although devoid of hexa-acylated species, B. mallei LPS appears to be a potent hTLR4 agonist and is, therefore, likely to play a significant role in the pathogenesis of human disease. TNF-a and IL-1b produced by LPS stimulated macrophages are two of the major pro-inflammatory cytokines responsible for the clinical manifestations of endotoxic shock. [unreadable] [unreadable] Previous studies have demonstrated that the O-antigens expressed by these organisms are required for serum resistance. More importantly, the O-antigens have been also been identified as putative vaccine candidates for immunoprophylaxis against melioidosis and glanders. Studies have shown that the O-antigens expressed by B. mallei isolates are antigenically and structurally similar to those expressed by B. pseudomallei isolates. Structural analyses by Burtnick et al have demonstrated that like the predominant O-antigen expressed by B. pseudomallei isolates, the O-antigen expressed by B. mallei is an unbranched heteropolymer consisting of disaccharide repeats having the structure 3)-beta-D-glucopyranose-(1-3)-6-deoxy-alpha-L-talopyranose-(1- in which the 6-deoxy-a-L-talopyranose (L-6dTalp) residues are non-stoichiometrically modified by 2-O-acetyl and 2-O-methyl substitutions. Unlike B. pseudomallei, however, the L-6dTalp residues of the B. mallei O-antigen do not appear to be acetylated at the O-4 position. In contrast to many other Gram-negative pathogens, virulent isolates of B. mallei and B. pseudomallei appear to express a very limited repertoire of chemically distinct O-antigens. Based upon these findings, we have recently initiated studies to develop melioidosis and glanders vaccine candidates utilizing O-antigens purified from B. pseudomallei and B. mallei isolates. [unreadable] [unreadable] Burkholderia - macrophage interactions:[unreadable] [unreadable] The study of pathogen host cell interactions in vitro is an important tool to define and characterize virulence factors of intracellular bacterial pathogens. The major species of Burkholderia include B. pseudomallei; B. mallei and an avirulent environmentally stable isolate B. thailandensis. B. pseudomallei macrophage interactions have been extensively studied but there is little known about the interactions of B. mallei with macrophages. We have performed a comparative analysis of B. mallei and B. pseudomallei macrophage interactions using the murine macrophage cell line (RAW 264.7). Our findings show that although B. mallei is capable of invading and replicating in RAW cells it is less efficiently internalized and grows more slowly. The optimal multiplicity of infection is critical for permissive B. mallei intracellular growth. In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells. Furthermore, treatment of activated macrophages with the iNOS inhibitor, aminoguanidine, inhibited clearance of B. mallei from infected monolayers. Based upon these results, it appears that MOIs significantly influence the outcome of interactions between B. mallei and murine macrophages and that iNOS activity is critical for the clearance of B. mallei from activated RAW 264.7 cells. We further tested differences in intracellular survival and multiplication among wild type and various mutants of B. mallei and B. pseudomallei. Eighteen mutants produced in each background of B. mallei and B. pseudomallei were tested in the RAW cell infection model. A type III secretion mutant of B. pseudomallei (strain 26bT3) showed marked differences in internalization and growth in RAW cells. An identical B. mallei type III secretion mutant (BMT3) and a B. mallei LPS mutant (GMrmlD) were incapable of growth in RAW cells. The results indicated that in vitro modeling of virulence using RAW macrophages is a simple and credible approach to screen Burkholderia mutants as a rational for analyses in animals.